Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.[Keywords: RNF20; BRE1; H2B ubiquitylation; tumor suppressor; transcription] Supplemental material is available at http://www.genesdev.org. Received June 6, 2008; revised version accepted August 12, 2008. Eukaryotic DNA is packaged into a chromatin structure of repeating nucleosomes consisting of DNA wrapped around an octamer of core histone proteins (H2A, H2B, H3, and H4). The histone tails, which protrude from the nucleosome, are subjected to a multitude of covalent modifications believed to play a vital role in chromatin remodeling and transcriptional regulation (Jenuwein and Allis 2001;Berger 2007;. One such modification is histone H2B monoubiquitylation. In the yeast S. cerevisiae this process is mediated by the E3 ligase BRE1 (Hwang et al. 2003). In mammals, the hBRE1(RNF20)/RNF40 complex was shown to function as the relevant E3 ligase (Kim et al. 2005;Zhu et al. 2005). In yeast, transcription of several inducible genes is impaired in the absence of ubiquitylated H2B (H2Bub) (Kao et al. 2004). Increased levels of H2Bub occur on the GAL1 core promoter and throughout the transcribed region upon transcriptional activation, with both ubiquitylation and deubiquitylation being required for optimal transcription (Henry et al. 2003;Xiao et al. 2005). Moreover, H2B monoubiquitylation was shown to lead to H3 methylation on Lys 4 and Lys 79, considered marks of actively transcribed genes (Briggs et al. 2002;Sun and Allis 2002). Yet, a recent study suggests that H2B ubiquitylation in S. pombe controls transcriptional elongation by RNA polymerase II (Pol II) independently of H3 methylation (Tanny et al. 2007).Along with the studies linking H2Bub positively with active transcription, other reports suggest a link between

The muscle specific ubiquitin E3 ligase MuRF1 has been implicated as a key regulator of muscle atrophy under a variety of conditions, such as during synthetic glucocorticoid treatment. FOXO class transcription factors have been proposed as important regulators of MuRF1 expression, but its regulation by glucocorticoids is not well understood. The MuRF1 promoter contains a near-perfect palindromic glucocorticoid response element (GRE) 200 base pairs upstream of the transcription start site. The GRE is highly conserved in the mouse, rat, and human genes along with a directly adjacent FOXO binding element (FBE). Transient transfection assays in HepG2 cells and C 2C12 myotubes demonstrate that the MuRF1 promoter is responsive to both the dexamethasone (DEX)-activated glucocorticoid receptor (GR) and FOXO1, whereas coexpression of GR and FOXO1 leads to a dramatic synergistic increase in reporter gene activity. Mutation of either the GRE or the FBE significantly impairs activation of the MuRF1 promoter. Consistent with these findings, DEXinduced upregulation of MuRF1 is significantly attenuated in mice expressing a homodimerization-deficient GR despite no effect on the degree of muscle loss in these mice vs. their wild-type counterparts. Finally, chromatin immunoprecipitation analysis reveals that both GR and FOXO1 bind to the endogenous MuRF1 promoter in C 2C12 myotubes, and IGF-I inhibition of DEX-induced MuRF1 expression correlates with the loss of FOXO1 binding. These findings present new insights into the role of the GR and FOXO family of transcription factors in the transcriptional regulation of the MuRF1 gene, a direct target of the GR in skeletal muscle.forkhead transcription factor class O; muscle RING finger 1; glucocorticoid receptor SKELETAL MUSCLE IS A DYNAMIC TISSUE that has the capacity to continuously regulate its size in response to a variety of external cues, including mechanical load, neural activity, hormones/growth factors, stress, and nutritional status. In addition, skeletal muscle serves as the most significant repository for protein in the body, a source that is tapped to provide a pool of amino acids for tissue repair and gluconeogenesis under conditions of starvation and other metabolic stresses. Muscle loss or "atrophy" occurs as the result of a number of disparate conditions, including aging, immobilization, metabolic diseases, cancer, and neurodegenerative diseases, and as a serious side effect of therapeutic corticosteroid hormone treatment (15,27,32). The recently identified E3 ubiquitin ligase, muscle RING finger 1 (MuRF1

Post-translational histone modifications have essential roles in controlling nuclear processes; however, the specific mechanisms regulating these modifications and their combinatorial activities remain elusive. Cyclin-dependent kinase 9 (CDK9) regulates gene expression by phosphorylating transcriptional regulatory proteins, including the RNA polymerase II carboxy-terminal domain. Here, we show that CDK9 activity is essential for maintaining global and gene-associated levels of histone H2B monoubiquitination (H2Bub1). Furthermore, CDK9 activity and H2Bub1 help to maintain correct replication-dependent histone messenger RNA (mRNA) 3 0 -end processing. CDK9 knockdown consistently resulted in inefficient recognition of the correct mRNA 3 0 -end cleavage site and led to increased read-through of RNA polymerase II to an alternative downstream polyadenylation signal. Thus, CDK9 acts to integrate phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing.

The estrogen receptor-a (ERa) determines the phenotype of breast cancers where it serves as a positive prognostic indicator. ERa is a well-established target for breast cancer therapy, but strategies to target its function remain of interest to address therapeutic resistance and further improve treatment. Recent findings indicate that proteasome inhibition can regulate estrogen-induced transcription, but how ERa function might be regulated was uncertain. In this study, we investigated the transcriptome-wide effects of the proteasome inhibitor bortezomib on estrogen-regulated transcription in MCF7 human breast cancer cells and showed that bortezomib caused a specific global decrease in estrogen-induced gene expression. This effect was specific because gene expression induced by the glucocorticoid receptor was unaffected by bortezomib. Surprisingly, we observed no changes in ERa recruitment or assembly of its transcriptional activation complex on ERa target genes. Instead, we found that proteasome inhibition caused a global decrease in histone H2B monoubiquitination (H2Bub1), leading to transcriptional elongation defects on estrogen target genes and to decreased chromatin dynamics overall. In confirming the functional significance of this link, we showed that RNA interference-mediated knockdown of the H2B ubiquitin ligase RNF40 decreased ERa-induced gene transcription. Surprisingly, RNF40 knockdown also supported estrogen-independent cell proliferation and activation of cell survival signaling pathways. Most importantly, we found that H2Bub1 levels decrease during tumor progression. H2Bub1 was abundant in normal mammary epithelium and benign breast tumors but absent in most malignant and metastatic breast cancers. Taken together, our findings show how ERa activity is blunted by bortezomib treatment as a result of reducing the downstream ubiquitin-dependent function of H2Bub1. In supporting a tumor suppressor role for H2Bub1 in breast cancer, our findings offer a rational basis to pursue H2Bub1-based therapies for future management of breast cancer. Cancer Res; 71(17); 5739-53. Ó2011 AACR.

SUMMARYThe estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target.